Morphological and Genetic Diversity of Opisthosporidia: New Aphelid Paraphelidium tribonemae gen. et sp. nov.

Aphelids are a poorly known group of parasitoids of algae that have raised considerable interest due to their pivotal phylogenetic position. Together with Cryptomycota and the highly derived Microsporidia, they have been recently re‐classified as the Opisthosporidia, which constitute the sister group to the fungi within the Holomycota. Despite their huge diversity, as revealed by molecular environmental studies, and their phylogenetic interest, only three genera have been described (Aphelidium, Amoeboaphelidium, and Pseudaphelidium), from which 18S rRNA gene sequences exist only for Amoeboaphelidium and Aphelidium species. Here, we describe the life cycle and ultrastructure of a new representative of Aphelida, Paraphelidium tribonemae gen. et sp. nov., and provide the first 18S rRNA gene sequence obtained for this genus. Molecular phylogenetic analysis indicates that Paraphelidium is distantly related to both Aphelidium and Amoebaphelidium, highlighting the wide genetic diversity of aphelids. Paraphelidium tribonemae has amoeboflagellate zoospores containing a lipid‐microbody complex, dictyosomes, and mitochondria with rhomboid cristae, which are also present in trophonts and plasmodia. The amoeboid trophont uses pseudopodia to feed from the host cytoplasm. Although genetically distinct, the genus Paraphelidium is morphologically indistinguishable from other aphelid genera and has zoospores able to produce lamellipodia with subfilopodia like those of Amoeboaphelidium.     

Functional responses of contrasting seed predator guilds to masting in two Mediterranean oak species

The predator satiation hypothesis poses that synchronous and variable seed production during masting events increases seed escape through seed predator satiation. The success of this strategy depends upon the type of consumer functional response, in this case defined as the change in seed consumption rate by a predator as a function of change in seed density. Type II (where the proportion of seed consumed is highest at low levels of seed availability) and type III (where the proportion of seed consumed is highest at some intermediate level of seed availability and then declines towards zero) functional responses describe negative density‐dependence and indicate predator satiation. The type of function response should be contingent upon herbivore traits: type II responses are predicted for dietary specialist predators with low mobility, and type III responses are predicted for highly mobile, dietary generalist predators. Surprisingly, most studies have not evaluated whether functional responses vary among seed predator guilds. Here we describe the functional responses at population and individual tree level of highly mobile generalist (birds and rodents) and less mobile specialist (insects) pre‐dispersal seed predators attacking acorns of two sympatric oaks (Quercus suber and Q. canariensis) over a 10‐year period. Our results showed that in most cases specialist seed predators exhibited the predicted type II functional response at both the individual tree and population level for both oak species. However, generalist seed predators did not exhibit the predicted type III response; instead, they also exhibited a type II response at the individual tree and population level for both oak species. By independently assessing the effects of multiple seed predators associated with the same host tree species, our work highlights the influence of herbivore traits on the outcome of plant–seed predator interactions in masting species, and thus furthers our understanding of the ecological and evolutionary mechanisms underlying masting behaviour.     

Antioxidant treatment reverses mitochondrial dysfunction in a sepsis animal model

Evidence from the literature has demonstrated that reactive oxygen species (ROS) play an important role in the development of multiple organ failure and septic shock. In addition, mitochondrial dysfunction has been implicated in the pathogenesis of multiple organ dysfunction syndrome (MODS). The hypothesis of cytopathic hypoxia postulates that impairment in mitochondrial oxidative phosphorylation reduces aerobic adenosine triphosphate (ATP) production and potentially induces MODS. In this work, our aim was to evaluate the effects of antioxidants on oxidative damage and energy metabolism parameters in liver of rats submitted to a cecal ligation puncture (CLP) model of sepsis. We speculate that CLP induces a sequence of events that culminate with liver cells death. We propose that mitochondrial superoxide production induces mitochondrial oxidative damage, leading to mitochondrial dysfunction, swelling and release of cytochrome c. These events occur in early sepsis development, as reported in the present work. Liver cells necrosis only occurs 24 h after CLP, but all other events occur earlier (6-12 h). Moreover, we showed that antioxidants may prevent oxidative damage and mitochondrial dysfunction in liver of rats after CLP. In another set of experiments, we verified that L-NAME administration did not reverse increase of superoxide anion production, TBARS formation, protein carbonylation, mitochondrial swelling, increased serum AST or inhibition on complex IV activity caused by CLP. Considering that this drug inhibits nitric oxide synthase and that no parameter was reversed by its administration, we suggest that all the events reported in this study are not mediated by nitric oxide. In conclusion, although it is difficult to extrapolate our findings to human, it is tempting to speculate that antioxidants may be used in the future in the treatment of this disease.     

ChromaPipe: a pipeline for analysis, quality control and management for a DNA sequencing facility

Optimizing and monitoring the data flow in high-throughput sequencing facilities is important for data input and output, for tracking the status of results for the users of the facility, and to guarantee a good, high-quality service. In a multi-user system environment with different throughputs, each user wants to access his/her data easily, track his/her sequencing history, analyze sequences and their quality, and apply some basic post-sequencing analysis, without the necessity of installing further software. Recently, Fiocruz established such a core facility as a "technological platform". Infrastructure includes a 48-capillary 3730 DNA Sequence Analyzer (Applied Biosystems) and supporting equipment. The service includes running samples for large-scale users, performing DNA sequencing reactions and runs for medium and small users, and participation in partial or full genome projects. We implemented a workflow that fulfills these requirements for small and high throughput users. Our implementation also includes the monitoring of data for continuous quality improvement (reports by plate, month and user) by the sequencing staff. For the user, different analyses of the chromatograms, such as visualization of good quality regions, as well as processing, such as comparisons or assemblies, are available. So far, 180 users have made use of the service, generating 155,000 sequences, 35% of which were produced for the BCG Moreau-RJ genome project. The pipeline (named ChromaPipe for Chromatogram Pipeline) is available for download by the scientific community at the url http://bioinfo.pdtis.fiocruz.br/ChromaPipe/. The support for assembly is also configured as a web service: http://bioinfo.pdtis.fiocruz.br/Assembly/.     

Ferric species of the giant extracellular hemoglobin of Glossoscolex paulistus as function of pH: an EPR study on the irreversibility of the heme transitions

The present article is focused on the transitions of ferric heme species of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) induced by successive alterations in pH, involving alkaline and acid mediums. Electron paramagnetic resonance (EPR) is the spectroscopy used to evaluate the transitions that occur in the first coordination sphere of ferric ion as a consequence of ligand changes in a wide range of pH, since this tool is very sensitive to slight changes that occur in the heme pocket of paramagnetic species. This approach is adequate to obtain information regarding the reversibility/irreversibility that involves the heme transitions induced by pH, since the degree of reversibility is associated to the intensity of the changes that occur in the spatial configuration of the polypeptide chains, which is clearly associated to the first coordination sphere. The results demonstrate a significant degree of irreversibility of heme transitions, since the final species, which do not present any change after 6 h of its respective formations, are quite different of the initial species. The results denote that the more stable species are the bis-histidine (hemichrome) and pentacoordinate species, due to the properties of their ligands and to the mechanical influence of the respective subunits. EPR spectra allow to distinguish the types of hemichrome species, depending on the reciprocal orientation between the histidine axial ligands, in agreement with Walker's Classification [Walker, F.A., 1999. Magnetic spectroscopic (EPR, ESEEM, M?ssbauer, MCD and NMR) studies of low-spin ferriheme centers and their corresponding heme proteins. Coord. Chem. Rev. 185-186, 471-534]. However, these transitions are not completed, i.e., the appearance of a determined species does not mean the total consumption of its precursor species, implying the coexistence of several types of species, depending on pH. Furthermore, it is possible to conclude that a "pure" EPR spectrum of aquomet ferric species is an important indicator of a high level of conservation referent to the "native" configuration of whole hemoglobin, which is only encountered at pH 7.0. The results allow to infer important physico-chemical properties as well as to evaluate aspects of the structure-activity relationship of this hemoprotein, furnishing information with respect to the denaturation mechanism induced by drastic changes in pH. These data are very useful since HbGp has been proposed as prototype of substitute of blood, thus requiring wide knowledge about its structural and chemical properties.     

The involvement of CD4+CD25+ T cells in the acute phase of Trypanosoma cruzi infection

The infection with Trypanosoma cruzi leads to a vigorous and apparently uncontrolled inflammatory response in the heart. Although the parasites trigger specific immune response, the infection is not completely cleared out, a phenomenon that in other parasitic infections has been attributed to CD4+CD25+ T cells (Tregs). Then, we examined the role of natural Tregs and its signaling through CD25 and GITR in the resistance against infection with T. cruzi. Mice were treated with mAb against CD25 and GITR and the parasitemia, mortality and heart pathology analyzed. First, we demonstrated that CD4+CD25+GITR+Foxp3+ T cells migrate to the heart of infected mice. The treatment with anti-CD25 or anti-GITR resulted in increased mortality of these infected animals. Moreover, the treatment with anti-GITR enhanced the myocarditis, with increased migration of CD4+, CD8+, and CCR5+ leukocytes, TNF-alpha production, and tissue parasitism, although it did not change the systemic nitric oxide synthesis. These data showed a limited role for CD25 signaling in controlling the inflammatory response during this protozoan infection. Also, the data suggested that signaling through GITR is determinant to control of the heart inflammation, parasite replication, and host resistance against the infection.     

Evaluation of the genotoxicity of piplartine, an alkamide of Piper tuberculatum, in yeast and mammalian V79 cells

The genus Piper belongs to the Piperaceae family, and includes species of commercial and medicinal importance. Chemical studies on Piper species resulted in the isolation of several biologically active molecules, including alkaloid amides, such as piplartine. This molecule, isolated from Piper tuberculatum, has significant cytotoxic activity against tumor cell lines, and presents antifungal, anti-platelet aggregation, anxiolytic, and antidepressant effects. In order to understand the biological properties of piplartine, this study investigated the genotoxicity and the induction of apoptosis by piplartine in V79 cells and its mutagenic and recombinogenic potential in Saccharomyces cerevisiae. Piplartine induced dose-dependent cytotoxicity in S. cerevisiae cultures in either stationary—or exponential growth phase. In addition, piplartine was not mutagenic when cells were treated during exponential-growth phase and kept in buffer solution, but it increased the frequencies of point, frameshift, and forward mutations when cells were treated in medium during growth. Piplartine treatment induced DNA strand breaks in V79 cells, as detected by neutral and alkaline comet assay. In cell cycle analysis, piplartine induced G2/M cell cycle arrest, probably as a consequence of the DNA damage induced and repair. Moreover, piplartine treatment induced apoptosis in a dose-dependent manner, as observed by a decrease in mitochondrial membrane potential and an increase in internucleosomal DNA fragmentation. These data suggest that the DNA damage caused by piplartine induces G2/M cell cycle arrest, followed by apoptosis. Moreover, we suggest that cells surviving piplartine-induced DNA damage can accumulate mutations, since this alkaloid was mutagenic and recombinogenic in S. cerevisiae assays.     

A metagenomic analysis of soil bacteria extends the diversity of quorum-quenching lactonases

A metagenomic library of 10 121 clones, generated from bacteria inhabiting a pasture soil from France, was screened for the presence of fosmids conferring either N -acylhomoserine lactone (NAHL) synthesis or NAHL degradation ability upon their Escherichia coli host. No clone producing NAHLs was identified whereas one, containing a 31 972 bp insert in fosmid p2H8, allowed NAHL degradation. This led to the cloning and identification of a gene, qlcA , encoding an NAHL-lactonase activity, as judged by lactone-ring closure and HPLC/MS analyses of NAHL degradation products. The qlcA gene efficiently quenched quorum-sensing regulated pathogenic functions when expressed in Pectobacterium carotovorum . The QlcA peptide belongs to the family of zinc-dependent metallohydrolases and appears to be distantly related to other NAHL-lactonases discovered in Agrobacterium , Bacillus , Photorhabdus and Rhizobium . In-silico analysis of the metagenomic insert revealed the occurrence of 20 orf , with a constant GC% and codon usage, suggesting a unique bacterial origin. Nine out of these 20 orf were homologous to genes encoding biosynthesis of arginine; they were clustered with an unusual succession argFJADBCRGH . The fosmid p2H8 is able to complement the argA , argB and argC mutants in E. coli . Phylogenetic analysis showed that 9 orf out of 20 were related to sequences from members of the Acidobacteria , supporting the hypothesis that the analysed insert might be originated from an organism related to this phylum.     

Tooth loss, denture wearing and associated factors among an elderly institutionalised Brazilian population

Objective: This cross‐sectional study assessed the prevalence and extent of tooth loss as well as denture wear and associated factors in institutionalised elderly in Brazil. Background: There is scarcity of research in Brazil concerning geriatric oral health issues. Material and methods: A sample of 335 individuals over 60 years of age, representative of the institutionalised elderly from Belo Horizonte, Brazil, were selected through a stratified sampling strategy. Data were collected from structured questionnaires, reviews of medical records and intra‐oral examinations. Results: Edentulousness was observed in 74.9% of the elderly and was related to age, years of study, income, length of institutionalisation, systemic diseases and mini‐mental score. Among the dentate subjects, 17.9% possessed 20 or more teeth. Approximately half the edentulous elderly (42.6%) and most of the partially dentate subjects (66.7%) did not wear dentures. The multivariate analysis showed that subjects that wear dentures were more likely to be female, possess higher incomes, be more functionally independent and married. Furthermore, the odds of wearing dentures among the elderly who had lost 13–32 teeth were 9.11 times higher than those who had lost up to 12 teeth. Conclusion: Tooth loss is highly prevalent in this institutionalised Brazilian population, and the prevalence of denture wear is low. The extent of tooth loss, income, functional status and marital status are important predictors for denture wear. The development of an oral health programme for these individuals should assist in their rehabilitation needs and promote in‐home care or safe transport for the elderly to a health care location.